The ability of an enzyme to discriminate among many potential substrates is an important factor in maintaining the fidelity of most biological functions. While substrate selection can be regulated on many levels in a biological context, such as spatial and temporal localization of enzyme and substrate, concentrations of enzyme and substrate, and requirement of cofactors, the substrate specificity at the enzyme active site is the overriding principle that determines the turnover of a substrate. Characterization of the substrate specificity of an enzyme clearly provides invaluable information for the dissection of complex biological pathways. Definition of substrate specificity also provides the basis for the design of selective substrates and inhibitors to study enzyme activity.
Of the genomes that have been completely sequenced, 2% of the gene products encode proteases (Barrett, A. J., et al., (1998) Handbook of Proteolytic Enzymes (Academic Press, London)). This family of enzymes is crucial to every aspect of life and death of an organism. With the identification of new proteases, there is a need for the development of rapid and general methods to determine protease substrate specificity. While several biological methods, such as peptides displayed on filamentous phage (Matthews, D. J., et al. (1993) Science 260:1113-7; Ding, L., et al., (1995) Proceedings of the National Academy of Sciences of the United States of America 92:7627-31), and chemical methods, such as support-bound combinatorial libraries (Lam, K. S., et al., (1998) Methods in Molecular Biology, 87:1-6), have been developed to identify proteolytic substrate specificity, few offer the ability to rapidly and continuously monitor proteolytic activity against complex mixtures of substrates in solution.
The use of 7-amino-4-methyl coumarin (AMC) fluorogenic peptide substrates is a well-established method for the determination of protease specificity (Zimmerman, M., et al., (1977) Analytical Biochemistry 78:47-51). Specific cleavage of the anilide bond liberates the fluorogenic AMC leaving group allowing for the simple determination of cleavage rates for individual substrates. More recently, arrays (Lee, D., et al., (1999) Bioorganic and Medicinal Chemistry Letters 9:1667-72) and positional-scanning libraries (Rano, T. A., et al., (1997) Chemistry and Biology 4:149-55) of AMC peptide substrate libraries have been employed to rapidly profile the N-terminal specificity of proteases by sampling a wide range of substrates in a single experiment. Each of these published efforts was designed for profiling caspases, cysteine proteases that require an Asp residue at the P1-position for substrate turnover. This requirement allows for the convenient attachment of the P1-Asp to the solid-support through the carboxylic acid side-chain. Since most proteases do not require P1-Asp/Glu for activity, libraries generated by these methods have limited applicability. Naturally, fluorogenic substrates that contain P1-amino acids that do not possess adequate side-chain functionality for attachment to a solid support in a straightforward manner (Gly, Leu, Val, Ile, Ala, Pro, Phe) will not be amenable to similar synthetic strategies.
Recently Fmoc-based synthesis methods to displace support-bound peptides with nucleophiles in a final cleavage step to produce C-terminal modified peptides have been developed (Backes et al., (1999) Journal of Organic Chemistry 64:2322-2330). The preparation of fluorogenic peptide substrates with any residue at the P1-position is possible by the preparation of AMC-amino acid derivatives, which are then used as nucleophiles to produce the AMC-peptide substrates (Backes et al. (2000) Nature Biotechnology 18(2): 187-193).
Support bound fluorogenic materials are also known in the art. For example, Adamczyk et al., Bioorg. Med. Chem. Lett., 9:217-220 (1999), have disclosed resin-supported fluorophores prepared from a new N-hydroxysuccinimidyl resin. The resin-bound active esters were used to prepare conjugates with haptens, such as estriol, thyroxine, phenytoin, etc. As the fluorophore is transferred from the resin to the free hapten, the resin-bound fluorophores of Adamczyk et al. do not constitute an appropriate starting point for the solid-phase synthesis of a peptide, nor is the use of the resin-bound fluorophore for derivatization of pre-formed peptides disclosed.
While the art provides a selection of methods that are useful for labeling materials with fluorophores, a method for the solid-phase synthesis of fluorogenic peptides, which begins with a resin-bound fluorophore, and materials that allow the method to be practiced, would represent a significant advance in the art. Such a method has great utility and provides a general strategy for the preparation of fluorogenic peptide substrate libraries. An innovative method would meet the following objectives: (1) the solid-phase synthesis method should enable direct incorporation of at least all 20 proteinogenic amino acids at every position, including the P1-position; (2) the method should be compatible with art-recognized solid-phase peptide synthesis protocols and instrumentation; and (3) the method should be flexible enough to enable the rapid synthesis of any single substrate, substrate array, and positional scanning library. Quite surprisingly, the present invention provides such a method.